Effects of prenatal exposure to extremely low electro-magnetic field on in vivo derived blastocysts of mice.

BACKGROUND
Indisputable population exposure to widespread electromagnetic fields, has grown concerns over the probable health effects of these fields.


OBJECTIVE
The present study was aimed to examine the possible effects of 50 Hz extremely low frequency electromagnetic field (ELF-EMF) exposure on the number and quality of mice blastocysts.


MATERIALS AND METHODS
In present study, 66 NMRI pregnant females divided into two treated and non-treated groups. The treated group exposed to ELF-EMF (50 Hz and 6×10(-3) T). Subsequently, embryos were collected by flushing the uterine horn and Fallopian tubes on the day 3 of gestation. Number of trophoectoderm (TE) and Inner Cell Mass (ICM) cells in blastocysts were determined after differential nuclei staining using a modified method. Furthermore, number of all flushed blastocysts calculated in each group.


RESULTS
There was no significant difference in mean number of blastocysts in treated (6.64±1.34) and none treated (8.22±1.59) groups. In treated group, there were significant decreased in total cell number of blastocysts (p=0.000), number of ICM cells (p=0.000), and number of TE cells (p=0.001) whereas the ratio of ICM/TE cells increased (p=0.002).


CONCLUSION
The data indicate that ELF-EMF is able to affect cellular composition of blastocysts, but it can't omit total volume of blastocysts.


Introduction
owadays, widespread applications of electrical instruments have resulted in population continuous exposure to electromagnetic fields (EMF), produced by electric current flows.
The probable effects of these fields, particularly on human health, have attracted researchers' attention to perform more investigations (1). Recent investigations have manifested that even small changes of EMF intensity can induce biological disorders (2). The most prominent EMFs expose that modern societies in ordinary life are Nonionizing 50-60 Hz ELF-EMFs (3).
Studies, assessing the reproductive influences of power frequency (50-60 Hz) exposure, demonstrated different and sometimes adverse results in males and females. Increased risk of miscarriage caused by ELF-EMF exposure during pregnancy in women, changes in uterus and ovary in rats, congenital deformities and developmental delay in the offspring of mice, increased height of mice fallopian tube epithelial cells and reduced fertility in female rats are some outcomes of these investigations in females (4-10). It has also been investigated that 50-Hz magnetic field reduces the secretion of hormones and increases the apoptosis-related gene expression in human villous trophoblasts in vitro (11,12).
Although, in recent years, many human diseases have been suspected to associate with ELF-EMF, by means of performing in vitro experiments, but there has not been any definitive evidence to prove these associations. Thus, ELF-EMF involvement in onset of disorders has remained somewhat controversial. Starting from these premises, present research aimed to judge the probable N effects of 50 Hz ELF-EMF, on the quality and features of mice blastocysts.

Animals
In this experimental study, sixty six fertilized female NMRI (Naval Medical Research Institute) mice (8-12 weeks of age) were subjected to the experiments according to moral code: 5-11-6-88 of Arak University of Medical Sciences. The mice were randomly divided into 2 groups (33 animals per group): Group I (non-treated group) was not exposed to ELF-EMF and Group II (treated group) was exposed to ELF-EMF for 48 hours. Embryos were obtained by flushing the uterine horn and fallopian tubes on the day 3 of gestation with CMRL 1066 culture medium (Gibco; 21530-076) with 1milli-mol/liter L-glutamine (Sigma; G7513) and 100mM sodium pyruvate (Sigma; P8574).

Exposure system
The ELF-MF used in the present study was produced by a pair of Helmholtz coils able to generate a highly homogeneous field (with homogeneity 5/1000) (13,14). To avoid changing in heat and electromagnetic field, hose water was passed around sinusoid. The characteristics of the system were as follows: (I) Power supply: 220 V in, 25 V out, permanent current intensity 3 Ampere.
(II) Multi-meter to control the intensity of the current entering the instrument.
(III) A 50 Hz sinusoidal oscillating ELF-MF was produced by a 380 round turn coil twisted around a cylinder (19 cm diameter and 15.5 cm length) and containing a chamber to house the mice in the center of the cylinder, where the maximum even ELF-MF (6±0.1 mT) and temperature (37±0.1 o C) was recorded.
(IV) A Teslameter (compensation-51662, sensitivity ≥0.1 mT) was used for precise measurement of magnetic field intensity in the chamber.

Differential staining of blastocysts
The numbers of blastocysts in two groups were counted (n=490.36±1.46). Then, twenty five blastocysts of each group were randomly selected. Trophoectoderm cells (TE) and cells of Inner Cell Mass (ICM) were counted after differential nuclei staining using a modified method of Piekos et al (15). Briefly, embryos were submitted to zona removal using Tyrods' solution (pH=2.2).
After fixing in absolute ethanol containing 22g/ml bisbenzimide (Sigma; B 2261) at 5 o C overnight, individual blastocysts were mounted in glycerol on microscopic slides and compressed manually before visualizing by epi-fluorescence (Nikon; 801) using Nikon filters; UV-2A and G-2A. Blue nuclei were considered as originating from the inner cells (ICM) and red-to-pink fluorescing nuclei as belonging to the outer cells (TE) (Figure 1).

Statistical analysis
In this study all of the parameters were stated as means±SEM. The number of blastocytes, ICM and TE cells and also the ratio of ICM/TE cells were determined repeatedly four times and the standard deviations were calculated. Statistical analysis was done by paired T-test using SPSS 16.0 for Windows XP (SPSS Inc., Chicago, IL). p<0.05 was considered significant.

Results
The mean number of blastocysts, total cells in each blastocyst, ICM cells, TE cells and also the ratio of ICM to TE cells are shown in table I. The data indicated that there was no significant difference in the numbers of blastocysts in two groups (p=0.07) ( Table I).
The information about the quality of obtained blastocysts from mice following maternal exposure to ELF-EMF, are showed in figure I and the determined data about quality in table I.
In treated group, there were significantly decreased in the mean numbers of ICM cells (p=0.00), TE cells (p=0.01) and of total cells (p=0.00) whereas the ratio of ICM cells to TE cells was significantly increased (p=0.02).

Discussion
The present study showed that exposure to ELF-EMF is not able to affect the number of blastocysts. This data is similar with Hannele et al that showed that in vitro exposure to 50 Hz magnetic field did not induce considerable difference in the number of mouse early blastocyst and late blastocysts (16).
In parallel, Pafkova et al found no significant alterations in embryogenesis of rat and chick embryos, exposed to 50 Hz EMF at 6 or 10 tesla x10 -6 of intensity. These results are in contrast with Borhani et al that indicated significant decrease in the mean number of mice blastocysts at the result of exposure to 50-Hz EMF. This controversy could be due to the duration of exposure that was 48 hours in this study while Borhani et al exposed pregnant mice for 12 days (17,18).
However, the study showed that numbers of ICM cells, TE cells and total cells in blastocysts of ELF-EMF exposed group were significantly decreased. These indisputable decreases in mentioned numbers, indicates growth depression inducement in ELF-EMF exposed group.
Thus, present study confirmed the cellular toxicity of prenatal exposure to ELF-EMF as the same as that for adult rats (7). Although the mechanisms by which ELF-MF induce cytototoxicity is not completely understood, apoptosis is considered as main cellular event by many authors. In vivo studies showed that 50 Hz 0.2, 3.2 or 6.4 mT for 2 weeks or 4 weeks blocked the cell cycle in S phase in testes of mice and 60 Hz 14 mT magnetic field for 8 weeks induced apoptosis in testicular germ cell in mice (19,20).
In vitro studies confirmed in vivo experiments that showed induction of apoptosis in K562 human leukemia cell line by ELF-MF (1 mT, 50 Hz), in isolated human osteoclasts by pulsed electromagnetic fields (7.5 Hz), in isolated liver cells and peripheral blood sampled from newborn mice by ELF (50 HZ), in human normal and cancer cells by timevarying magnetic fields (60-Hz 6 mT), and also induction of micronuclei in rat bone marrow by (910-MHz) (21)(22)(23)(24)(25). In sub cellular level, regulations of many molecules were considered for induction of apoptosis by ELF-MF. The studies suggest formation of free radicals such as reactive oxygen species (ROS), heat-shock protein 70 (hsp70), cyclic adenosine mono phosphate (cAMP) and also P53 gene and its related gene; P38 and other regulating genes of apoptosis such as bcl-2 and bax (21,24,(26)(27)(28)(29)(30).
In conclusion, this study indicated that exposure to ELF-EMF influence female mice fertility by affecting cellular composition of blastocysts. Equal number of blastocysts in treated and non-treated indicated that cytotoxic mechanisms such as apoptosis could not be enough to omit total volume of blastocysts.